Part of the work performed during development of this invention was supported by U.S. Government funds. The U.S. Government may have certain rights in this invention.
1. Field of the Invention
This invention relates to a gene therapy method for inducing pulmonary vasodilation by transducing a nitric oxide synthase gene into lung tissue. This invention also relates to methods of treating pulmonary hypertension and pharmaceutical compositions for treating pulmonary hypertension.
2. Related Art
Blood flow through the pulmonary circulation is highly regulated. For example, the pulmonary endothelium regulates pulmonary blood flow and maintains a low vascular resistance by releasing vasoactive substances, which control vasomotor tone, vascular patency, and normal vessel wall architecture. Vanhoutte, N. Engl. J. Med. 319:512-513 (1988).1 Vasomotor tone relates to the degree of active tension in the vessel wall and partially determines the luminal diameter of the vessel. Vascular patency refers to the condition of a blood vessel where the internal luminal diameter is normal and blood flow is unimpeded.
1 This article and all other articles, patents, or other documents cited or referred to in this application are specifically incorporated herein by reference. 
Nitric oxide is one compound that plays an important role in regulating pulmonary blood flow. However, it is a gas with no known storage mechanism, which diffuses freely across membranes and is extremely labile. Nitric oxide has a biological half-life on the order of seconds, and its production is tightly regulated.
Nitric oxide is produced by two classes of nitric oxide synthases (NOS). Nathan, FASEB J. 6:3051-3064 (1992). The constitutively expressed nitric oxide synthases, exist as two isoforms: the endothelial nitric oxide synthase (ceNOS) and the neuronal nitric oxide synthase, (nNOS). These isoforms are expressed in vascular endothelial cells, platelets, and in neural tissues such as the brain. This class of nitric oxide synthase is calcium and calmodulin dependent. In blood vessels ceNOS mediates endothelium dependent vasodilation in response to acetylcholine, bradykinin, and other mediators. Nitric oxide levels increase in response to shear stress, i.e., forces on the blood vessels in the direction of blood flow, and the mediators of inflammation. Furchgott and Vanhoutte, FASEB J. 3:2007-2018 (1989); Ignarro, FASEB J. 3:31-36 (1989).
In the nervous system, the neuronal NOS isoform is localized to discrete populations of neurons in the cerebellum, olfactory bulb, hippocampus, corpus striatum, basal forebrain, and brain stem. Bredt et al., Nature 347:768-770 (1990). Neuronal NOS is also concentrated in the posterior pituitary gland, in the superoptic and paraventricular hypothalmic nuclei, and in discrete ganglion cells of the adrenal medulla Id. The widespread cellular localization of the neuronal NOS isoform and the short half-life and diffusion properties of nitric oxide suggest that NOS plays a role in nervous system morphogenesis and synaptic plasticity.
The second class, inducible nitric oxide synthase (iNOS), is expressed in macrophages, hepatocytes, and tumor cells. Steuhr et al., Adv. Enzymol. Relat. Areas Mol. Biol. 65:287-346 (1992); Lowenstein et al., Proc. Natl. Acad. Sci. (USA) 89:6711-6715 (1992). The inducible form of NOS is not calcium regulated, but its expression is induced by cytokines. This form of NOS functions as a cytotoxic agent, and NO produced by inducible NOS targets tumor cells and pathogens. Hibbs et al., Biochem. Biophys. Res. Comm. 157:87-94 (1988); Nathan, FASEB J. 6:3051-3064 (1992); Marletta, Trends Biochem. Sci. 14:488-492 (1989).
All isoforms of NOS catalyze the conversion of L-arginine to L-citrulline with production of NO. In vascular smooth muscle cells and in platelets, NO activates soluble guanylate cyclase, which increases intracellular guanosine 3xe2x80x2,5xe2x80x2-cyclic monophosphate (cGMP), thereby inducing vasorelaxation and inhibiting platelet aggregation. The anti-platelet effect of NO and its vasodilatory and anti-proliferative action on pulmonary vascular smooth muscle cells suggest that NO may be an important modulator of pulmonary hypertension. Moncada et al., Pharmacol. Res. 43:109-142 (1991); Garg et al., J. Clin. Invest. 83:1774-1777 (1989); Roberts et al., Circ. Res. 76:215-222 (1995); Heath, Eur. Respir. Rev. 3:555-558 (1993); Radomski et al., Biochem. Biophys. Res. Commum. 148:1482-1489 (1987); Assender et al., J. Cardiovasc. Pharmacol. 17:104-107 (1991); de Graaf et al., Circulation 85:2284-2290 (1992).
The sequence of the various NOS isoforms have been published or are available in Genbank under the following accession numbers:
Each of these sequences are expressly incorporated herein by reference. The different forms of NOS are about 50 to 60 percent homologous overall.
Several in vitro and in vivo results suggest that NO may play a role in the pulmonary vascular response to hypoxia. For example, in perfused isolated lungs, hypoxia induces a significant reduction in contractile responses to acetylcholine and to inhibitors of NOS. Adnot et al., J. Clin. Invest. 87:155-162 (1991). In isolated pulmonary vascular rings hypoxia suppresses basal and agonist-stimulated release of NO. Johns et al., Circ. Res. 65:1508-1515 (1989); Shaul et al., J. Cardiovasc. Pharmacol. 22:819-827 (1993). In endothelial cells, hypoxia inhibits NO production by reducing ceNOS mRNA levels and ceNOS mRNA stability. McQuillan et al., Am J. Physiol. 267:H1921-H1927 (1994). Moreover, downregulation of ceNOS mRNA and protein correlate inversely with the severity of the plexogenic pulmonary arteriopathy in the lungs of patients with pulmonary hypertension. Giaid et al., N. Engl. J. Med. 333:214-221 (1995). Therefore, hypoxia-induced hypertension may correlate with reduced NO generation from pulmonary endothelium affecting the balance between pulmonary vasoconstrictive and vasodilatory stimuli.
In addition to hypoxia-induced pulmonary hypertension, there are other forms of pulmonary hypertension. For example, pulmonary hypertension can result from disease states such as interstitial lung diseases with fibrosis, e.g., sarcoidosis and pneumoconioses, e.g., silicosis. Pulmonary hypertension on can also result from emboli, from parasitic diseases such as schistosomiasis or filariosis, from multiple pulmonary artery thromboses associated with sickle cell disease, and from cardiac disease, such as cor pulmonale, and from ischemic and valvular heart disease.
In addition to resulting from other disease, pulmonary hypertension can also be a primary disease condition. Primary pulmonary hypertension is an uncommon disease, which can only be diagnosed after a thorough search for the usual causes of pulmonary hypertension. Ordinarily, the natural course of this disease encompasses about five years, and it is normally fatal, with treatment being palliative. While pharmacological vasodilator therapy for primary and secondary pulmonary hypertension is known, these methods often have undesirable systemic hypotensive side effects.
The use of gene therapy for the treatment of various diseases and disorders has advanced significantly over the last several years. In contrast to traditional pharmaceuticals, gene therapy refers to the transfer and insertion of new genetic information into cells or the substitution of deficient genetic information for the therapeutic treatment of diseases or disorders. In some cases the gene is expressed in the target cell, while in other cases expression is not required, e.g., antisense technology. The foreign gene is normally transferred into a cell that proliferates to spread the new gene throughout the cell population. Often stem cells or pluripotent progenitor cells are the target of gene transfer since they proliferate to various progeny lineages that may express the foreign gene.
High efficiency gene transfer systems for hematopoietic progenitor cell transformation have been described. See Morrow, Ann. N.Y. Acad Sci. 265:13 (1976); Salzar et al., In Organization and Expression of Globin Genes, A. R. Liss, Inc., New York at 313; Bernstein, In Genetic Engineering: Principles and Methods, Plenum Press, NY at 235; Dick et al., Trends in Genetics 2:165 (1986); Kiem, Curr. Opin. Oncol. 7;107-114 (1995). Viral vector transfer systems, such as retrovirus and adenovirus vectors, generally show a higher efficiency of transformation than DNA-mediated gene transfer procedures, such as Ca3(PO4)2 precipitation and DEAE dextran. Retroviral vector transfer systems also have the capacity to integrate transferred genes stably into a wide variety of cell types. However, retroviruses require proliferation of target cells for the expression of the newly transferred gene. Other non-vial methods of gene transfer include microinjection, eletoporation, liposomes, chromosome transfer, and transfection techniques. See Cline, Pharmacol. Ther. 29:69-92 (1985). However, these non-viral vectors have a relatively low in vivo transduction efficiency.
Therefore, there exists a need in the art to develop gene therapy methods to induce vasodilation in the pulmonary circulation and to treat pulmonary hypertension.
This invention satisfies these needs in the art by providing a method of inducing pulmonary vasodilation comprising introducing a vector containing a nitric oxide synthase gene operably linked to an expression control element into the lungs of a patient in need of pulmonary vasodilation. The nitric oxide synthase can be a constitutively expressed or an inducible nitric oxide synthase gene. In specific embodiments of this invention, the pulmonary vasodilation is selective, the vector is an adenovirus vector, the nitric oxide synthase gene is the endothelial nitric oxide synthase gene, and this vector is transduced into lung tissue as an aerosol. In more specific embodiments, the resulting pulmonary vasodilation does not significantly affect systemic blood pressure or cardiac index.
This invention also relates to a method of treating pulmonary hypertension comprising overexpressing nitric oxide synthase in the lungs of a patient in need of treatment by introducing the nitric oxide synthase gene operably linked to an expression control element into the lungs of a patient in need of treatment. In specific embodiments, this method can be used to treat hypoxia-induced pulmonary hypertension, primary pulmonary hypertension, and pulmonary hypertension secondary to pulmonary or cardiac disease states.
This invention further relates to a pharmaceutical composition comprising the nitric oxide synthase gene operably linked to an expression control element and a means for transducing said gene into pulmonary tissue. In an exemplary embodiment, the pharmaceutical composition comprises AdCMVceNOS in admixture with a pharmaceutically acceptable carrier.
Further features, objects and advantages of the present invention will become more fully apparent to one of ordinary skill in the art from a detailed consideration of the following description of the invention when taken together with the accompanying drawings.